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Friday, October 29, 2004

Antiseptics and Antibiotics (Kirby-Bauer test)

Antibiotics are defined as a chemical product that can inhibit or destroy pathogenic microorganisms.
Antiseptics are defined as chemicals that are able to inhibit in vitro sepsis. They do not kill the sepsis-producing agent, but inhibit its growth. Antiseptics are nontoxic to allow application to the skin and mucous membranes, such as Listerine.
The mode of action for these chemicals is the active denaturing of proteins within pathogenic organisms, cytoplasmic membrane dissolution, and act as oxidizing agents.
Clinical laboratories can test the potency of antibiotics and antiseptics by using the Kirby-Bauer test.A second example on this page includes a table for interpreting results according to critical diameters. You can determine from this table, assuming the test was done properly, if the bacterial agent is resistant, intermediate, or susceptible to the antibiotic that is being questioned.
Mueller-Hinton agar is the usual agar of choice for this procedure.
This test should be monitored closely, especially in the professional laboratory. Time limits and culture inoculum sizes should be within requirements for proper results. (Collegiate labs use a less restrictive Kirby-Bauer method).

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Thursday, October 21, 2004

Streptomyces


Streptomyces can form unique spores even in the presence of numerous antibiotics. They are the most successful bacterium in the Order Actinomycetales. They are found worldwide in soil and are important in soil ecology. In fact, much of the earthly smell of soil are given off by the Strptomyces species. They are also found in the medical industry because they synthesize antibiotics. They can eat almost anything, including sugars, alcohols, amino acids, and organic acids.
Click here to watch a cool clip!
If you don't have the required Quick Time Player, you can download it here.
Work cited: University of Leicester- Department of Microbiology and Immunology



Monday, October 18, 2004

Choosing the appropriate agar

Agar (or medium) is a gel-like polysaccharide used as a thickening agent to grow microorganisms. There are several types for different uses.

Complex- Has a variety of ingredients.
Defined- Pure chemicals within the mixture. They also need a buffer added to them.(Good source for anti-microbial requirements, AKA growth factors)
Selective - Helps to narrow down organism of interest. EX: Glucose salts.
Thayer-Martin - Isolates N.Gonorrhea, Antibiotics will inhibit growth of fungi, Gram positive and Gram negative rods, and will allow growth with little competition.
Differential- Created in mind that different organisms metabolize different materials in different ways. A bacteria type can change a substance in a recognizable way. EX: Blood agar- "strep test"
Glucose Salts - Only organisms that can make all cellular components from glucose and inorganic salts are able to grow.
Trypicase-Soy - Organisms must require vitamins and growth factors in order to grow on this agar.
EMB (Eosin Methylene Blue) - A selective medium that permits Gram-negative rods to grow, but inhibits Gram-positive bacterial growth.

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Special Topics in Gram Staining

Although the Gram-Stain process is outlined in a link below, this is an area describing the outcome of each step. (reference of Robert Bauman).

1) Flood the smear with Crystal Violet dye and then rinse with water. This primary stain colors all cells.
2) Flood the smear with Iodine solution for 1 min., then rinse with water. Iodine binds to a dye and makes it less soluble in water. All of the cells should be purple after this step.
3) Rinse with solution of ehanol and acetone for 30 sec., then rinse with water. This decolorizing agent breaks down thin cell walls that the Gram-negative cells have. These cells are now colorless, while the Gram-positive cells remain purple.
4) Flood the smear with safranin for 1 min. and then rinse with water. This red counterstain gives contrasting color to the primary stain. The slide is then blotted dry for microscopy. The thicker cell walls of the Gram-positives remain purple, while the Gram-negative cells are now pink.

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Classification of Flagella

Flagella are long, whiplike shafts that extend from the cellular body.
There are four basic types: Monotrichous, Lophotrichous, Amphitrichous, and Peritrichous.
Here's a view of what they look like.
The fifth and unusual type is atrichous, meaning without flagella.

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Sunday, October 17, 2004

Quantification of Microorganisms

The counting of bacteria is important if you want to know the number of bacteria in a sample. The common methods are: Plate count, Direct count, and Turbidometric

Plate count- is used on the premise that each viable bacterium will produce a colony when growing on a agar plate. A sample of the material to be counted is suspended in liquid and placed in a empty petri plate. Next, melted agar is poured into the plate. After incubation, each organism produces a colony in the agar that can be counted.
There are two main advantages of the plate count over other methods. Only viable organisms are counted, which are the ones considered to be important. Samples with small methods can also be counted.
The disadvantages are related to size and frequency. Bacteria are usually present in large numbers. E.coli could easily contain over one billion cells/ml. Some bacteria will stick together, gving rise that two different organisms may produce the appearance of only one colony.
Direct count - Organisms in a suspension of bacteria are placed on a slide that has been ruled into squares and can hold a specific amount of volume. By counting the bacteria that appear on the grid areas, the number of organisms in a sample can be calculated.
The direct count is much faster than the plate count but has its disadvantages. There must be a certain multiple number of organisms before there are enough to be seen, and both viable and nonviable organisms appear the same under the microscope.
Turbidometric - turbid simply means cloudy. In this method, a spectrophotometer measures the turbidity on bacteria in a broth. The more bacteria present, the cloudier the broth.

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Aerobic and Anaerobic Growth

Reference Material - Nester et al. Microbiology: A Human Perspective, 4th edition, 2004

DEFINITIONS:
Aerobic - In the presece of air.
Anaerobic - In the absence of air.
Agar deep - A test tube with agar filled near the top.

The appearance of aerobic and anaerobic growth in shake tubes in solidified agar appear differently.
In an Obligate aerobe, there is only surface growth above the agar, thus, needing air in order to grow.
In a Strict anaerobe, there is growth only at the bottom of the agar and no surface growth. These organisms do not need air in order to grow.
In a Facultative anaerobe tube, there is both surface growth and growth at the bottom of the agar. These organisms do not have air requirements and can grow with or without oxygen.

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Saturday, October 16, 2004

Staining

DEFINITIONS:

Staining - refers to simply coloring a specimen with a colored dye.

Simple Stains - using a simple basic dye, such as crystal violet, in order to view the shape , size, and arrangement of cells. The simple stains include: differential, Gram, acid-fast, and endospore stains
Multiple and Differential Stains - The most popular stain used in microscopy. These stains involve multiple dyes in order to distinguish cells under the microscope.
Gram Stain - Named after Hans Christian Gram, differenciates between Gram-positive purple and Gram-negative pink stains and is used to identify certain pathogens.
[Visit this page to watch the Gram-stain procedure.]
Special Stains - These are stains that include the acid-fast, endospore, capsule and flagellar stains.
Acid-Fast Stain - is used for staining cells of the genera Mycobacterium and Nocardia. These cells have a waxy material that repel the water-based dyes of the Gram Stain. The Mycobacterium and Nocardia cause many human diseases, such as TB, leprocy, and other pulmonary and skin infections.
[scroll halfway down this page and watch the acid-fast stain procedure.]
Endospore Stain - The cell walls of endospores are impermeable to most chemicals, and being in the genera Bacillus and Clostridium, cause diseases such as anthrax, teatanus and gangrene.
The staining process involves both a primary stain and a counterstain.
Capsule Stain - a stain used to reveal negatively charged bacterial capsules. The encapsulated cells will have a halo appearance under the microscope.
Flagellar Stain - Flagella are usually invisible under light microscopy, but their identification and anatomy are important in determining some pathogens. Certain chemicals that bind to the flagella are used in the staining process. The flagella color may change or an increase in contrast should make them visible.
Agar - a gel-like polysaccharide isolated from red algae and used as a thickening agent.
MEDIA TYPES:
Defined - is used when exact chemical composition is known. Pure chemicals are in the mixture and need a buffer added to them. These types are good for looking at nutritional requirements or growth factors.
Selective - contains substances that either favor the growth of some organisms or inhibit the growth of unwanted ones. This helps to narrow down the microorganism of interest. (Glucose salts)
Differential - created in mind that different organisms metabolize different materials in different ways. A bacteria type can change a substance in a recognizable way. (Blood agar used for strep testing)
Complex - these agars contain a variety of ingredients.
COLONIES:
A bacterial colony
A fungal colony


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Friday, October 15, 2004

Procedures

Simple, Gram, and Acid-fast staining procedures.

Endospore staining procedure

Calpuse staining procedure



Lab links:
Austin Community College
University of Leicester

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